The invention relates to a method for inserting genes into the chromosome of bacterial strains, and the resulting strains. In the biotech industry it is desirable to construct polypeptide production strains having several copies of a gene of interest stably chromosomally integrated, without leaving antibiotic resistance marker genes in the strains.
In the industrial production of polypeptides it is of interest to achieve a product yield as high as possible. One way to increase the yield is to increase the copy number of a gene encoding a polypeptide of interest. This can be done by placing the gene on a high copy number plasmid, however plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells. Another way to increase the copy number of the gene of interest is to integrate it into the host cell chromosome in multiple copies. It has previously been described how to integrate a gene into the chromosome by double homologous recombination without using antibiotic markers (Hone et al., Microbial Pathogenesis, 1988, 5: 407-418); integration of two genes has also been described (Novo Nordisk: WO 91/09129 and WO 94/14968). A problem with integrating several copies of a gene into the chromosome of a host cell is instability. Due to the sequence identity of the copies there is a high tendency for the them to recombine out of the chromosome again during cultivation of the host cell unless a selective marker or other essential DNA is included between the copies and selective pressure is applied during cultivation, especially if the genes are located in relative close vicinity of each other. It has been described how to integrate two genes closely spaced in anti-parallel tandem to achieve better stability (Novo Nordisk: WO 99/41358).
The present day public debate concerning the industrial use of recombinant DNA technology has raised some questions and concern about the use of antibiotic marker genes. Antibiotic marker genes are traditionally used as a means to select for strains carrying multiple copies of both the marker genes and an accompanying expression cassette coding for a polypeptide of industrial interest. In order to comply with the current demand for recombinant production host strains devoid of antibiotic markers, we have looked for possible alternatives to the present technology that will allow substitution of the antibiotic markers we use today with non-antibiotic marker genes. Thus in order to provide recombinant production strains devoid of antibiotic resistance markers, it remains of industrial interest to find new methods to stably integrate genes in multiple copies into host cell chromosomes.
The present invention solves the problem of integrating multiple copies of a gene of interest by homologous recombination into well defined chromosomal positions of a bacterial host strain which already comprises at least one copy of the gene of interest in a different position. This can be done by making a deletion of part of one or more conditionally essential gene(s) (hereafter called the xe2x80x9cintegration genexe2x80x9d) in the host chromosome of a strain which already comprises at least one copy of a gene of interest, or by otherwise altering the gene(s) to render it non-functional; or by integrating at least one partial non-functional conditionally essential gene into the host chromosome, so that the resulting strain has a deficiency (e.g. specific carbon-source utilization) or a growth requirement (e.g. amino acid auxotrophy) or is sensitive to a given stress. The next (i.e. second or third etc.) copy of the gene of interest is then introduced on a vector, on which the gene is flanked upstream by a partial fragment of the integration gene, and downstream is flanked by a fragment homologous to a DNA sequence downstream of the integration gene on the host chromosome. Thus, neither host chromosome nor the incoming vector contain a full version of the integration gene. In a non-limiting example the host chromosome may comprise the first two thirds of the integration gene and the vector the last two thirds, effectively establishing a sequence overlap of one third of the integration gene on the vector and the chromosome.
Expression of the full version of the integration gene will only occur if homologous recombination between vector and host chromosome takes place via the partial integration gene sequences, and this particular recombination event can be efficiently selected for, even against the background of homologous integration into the chromosome directed by the gene of interest into the identical gene(s) comprised on the chromosome already.
This strategy will enable directed gene integration by homologous recombination at predetermined loci, even though extended homology exists between the gene of interest on the incoming vector and other copies of this gene at other locations in the chromosome, and even though it is not feasible to identify the desired integrants based on the qualitative phenotype resulting from expression of the gene of interest, as this gene is already present in one or more copies in the host.
In a non-limiting example herein a Bacillus enzyme production strain is provided that comprises two anti-parallel copies (inverted orientation) of a gene encoding the commercially available amylase Termamyl(copyright) (Novo Nordisk, Denmark). A gene homologous to the dal gene of Bacillus subtilis, encoding a D-alanine racemase, was identified in the Bacillus production strain, it was sequenced and a partial deletion was made in the dal gene of the Bacillus two-copy Termamyl(copyright) strain. A vector was constructed to effect a stable non-tandem chromosomal insertion of a third Termamyl(copyright) gene copy adjacent to the dal locus, in the process effectively restoring the complete dal gene, according to the above strategy.
In another non-limiting example herein, an additional copy of the amylase encoding gene was introduced into the xylose isomerase operon of the Bacillus enzyme production strain which already comprised at least two copies of the amylase gene located elsewhere on the chromosome.
Also in a non-limiting example we demonstrate the method of the invention by integrating an additional amylase-encoding gene into the gluconat operon of the Bacillus enzyme production strain. Other non-limiting examples of integration into conditionally essential genes are given below.
Accordingly in a first aspect the invention relates to a method for constructing a cell comprising at least two copies of a gene of interest stably integrated into the chromosome in different positions, the method comprising the steps of:
a) providing a host cell comprising at least one chromosomal copy of the gene of interest, and comprising one or more conditionally essential chromosomal gene(s) which has been altered to render the gene(s) non-functional;
b) providing a DNA construct comprising:
i) an altered non-functional copy of the conditionally essential gene(s) of step a); and
ii) at least one copy of the gene of interest flanked on one side by i) and on the other side by a DNA fragment homologous to a host cell DNA sequence located on the host cell chromosome adjacent to the gene(s) of step a); wherein a first recombination between the altered copy of i) and the altered chromomosomal gene(s) of step a) restores the conditionally essential chromosomal gene(s) to functionality and renders the cell selectable;
c) introducing the DNA construct into the host cell and cultivating the cell under selective conditions that require a functional conditionally essential gene(s); and
d) selecting a host cell that grows under the selective conditions of the previous step; wherein the at least one copy of the gene of interest has integrated into the host cell chromosome adjacent to the gene(s) of step a); and optionally
e) repeating steps a) to d) at least once using a different chromosomal gene(s) in step a) in each repeat.
Another way of describing the first aspect of the invention relates to a method for constructing a cell comprising at least two copies of a gene of interest stably integrated into the chromosome in different positions, the method comprising the steps of:
a) providing a host cell comprising at least one chromosomal copy of the gene of interest;
b) altering a conditionally essential chromosomal gene(s) of the host cell whereby the gene becomes non-functional;
c) making a DNA construct comprising:
i) an altered non-functional copy of the chromosomal gene(s) of step b); and
ii) at least one copy of the gene of interest flanked on one side by i) and on the other side by a DNA fragment homologous to a host cell DNA sequence adjacent to the gene(s) of step b); wherein a first recombination between the altered copy of i) and the altered chromomosomal gene(s) of step b) restores the chromosomal gene(s) to functionality and renders the cell selectable;
d) introducing the DNA construct into the host cell and cultivating the cell under selective conditions that require a functional gene(s) of step b); and
e) selecting a host cell that grows under the selective conditions of step d); wherein the at least one copy of the gene of interest has integrated into the host cell chromosome adjacent to the gene(s) of step b); and optionally
f) repeating steps a) to e) at least once using a different chromosomal gene(s) in step b) in each repeat.
Herein genetic tools are also described in the form of DNA constructs necessary for carrying out the method of the invention.
Consequently in a second aspect the invention relates to a DNA construct comprising:
i) an altered non-functional copy of a conditionally essential chromosomal gene(s) from a host cell, preferably the copy is partially deleted; and
ii) at least one copy of a gene of interest flanked on one side by i) and on the other side by a DNA fragment homologous to a host cell DNA sequence located on the host cell chromosome adjacent to the conditionally essential gene(s) of i).
The present invention provides a method for obtaining a host cell comprising at least two copies of a gene of interest stably integrated on the chromosome adjacent to conditionally essential loci.
Accordingly in a third aspect the invention relates to a host cell comprising at least two copies of a gene of interest stably integrated into the chromosome, where at least one copy is integrated adjacent to a conditionally essential locus and wherein the cell is obtainable by any of the methods defined in the first aspects.
Another way of describing an aspect of the invention relates to a host cell comprising at least two copies of a gene of interest stably integrated into the chromosome, where each copy is integrated adjacent to different conditionally essential loci and wherein the cell is obtainable by any of the methods defined in the first aspects.
The method of the invention relies on complementing a conditionally essential gene(s) that was rendered non-functional, and a number of suitable host cells comprising such non-functional genes are described herein. To carry out multiple rounds of gene integration according to the invention it is advantageous to provide a host cell comprising several non-functional conditionally essential genes.
In a fourth aspect the invention relates to a Bacillus licheniformis cell, wherein at least two conditionally essential genes are rendered non-functional, preferably the genes are chosen from the group consisting of xylA, galE, gntK, gntP, glpP, glpF, glpK, glpD, araA, metC, lysA, and dal.
Any host cell as described herein for use in a method of the invention is intended to be encompassed by the scope of the invention.
Another aspect of the invention relates to the use of a cell as defined in the previous aspect in a method as defined in the first aspects.
As mentioned above, genetic tools of the invention are described herein, and it is intended that the scope of the invention comprises such constructs when present in or propagated in host cells as is common in the art.
Yet another aspect of the invention relates to a cell comprising a DNA construct as defined in the second aspect.
In a final aspect the invention relates to a process for producing an enzyme of interest, comprising cultivating a cell as defined in any of the preceding aspects under conditions appropriate for producing the enzyme, and optionally purifying the enzyme.